WDR36 inhibits the osteogenic differentiation and migration of periodontal ligament stem cells

Abstract

BACKGROUND

Periodontitis is an inflammatory disease caused by the host’s immune response and various interactions between pathogens, which lead to the loss of connective tissue and bone. In recent years, mesenchymal stem cell (SC) transplantation technology has become a research hotspot, which can form periodontal ligament, cementum, and alveolar bone through proliferation and differentiation.

AIM

To elucidate the regulatory effects of WD repeat-containing protein 36 (WDR36) on the senescence, migration, and osteogenic differentiation of periodontal ligament SCs (PDLSCs).

METHODS

The migration and chemotaxis of PDLSCs were detected by the scratch-wound migration test and transwell chemotaxis test. Alkaline phosphatase (ALP) activity, Alizarin red staining, calcium content, and real-time reverse transcription polymerase chain reaction (RT-qPCR) of key transcription factors were used to detect the osteogenic differentiation function of PDLSCs. Cell senescence was determined by senescence-associated β-galactosidase staining.

RESULTS

The 24-hour and 48-hour scratch-wound migration test and 48-hour transwell chemotaxis test showed that overexpression of WDR36 inhibited the migration/chemotaxis of PDLSCs. Simultaneously, WDR36 depletion promoted the migration/chemotaxis of PDLSCs. The results of ALP activity, Alizarin red staining, calcium content, and RT-qPCR showed that overexpression of WDR36 inhibited the osteogenic differentiation of PDLSCs, and WDR36 depletion promoted the osteogenic differentiation of PDLSCs. Senescence-associated β-galactosidase staining showed that 0.1 μg/mL icariin (ICA) and overexpression of WDR36 inhibited the senescence of PDLSCs, and WDR36 depletion promoted the osteogenic differentiation of PDLSCs.

CONCLUSION

WDR36 inhibits the migration and chemotaxis, osteogenic differentiation, and senescence of PDLSCs; 0.1 μg/mL ICA inhibits the senescence of PDLSCs. Therefore, WDR36 might serve as a target for periodontal tissue regeneration and the treatment of periodontitis.

Key Words: Periodontal ligament stem cells; Periodontal tissue regeneration; Icariin; WD repeat-containing protein 36; Osteogenic differentiation

Core Tip: Periodontal ligament stem cells (PDLSCs) are an important cell source for periodontal tissue regeneration. They can migrate to injured areas to promote tissue repair, induce osteogenic differentiation for bone tissue regeneration, and delay tissue loss by inhibiting senescence. In this study, we investigated the regulatory function of WD repeat-containing protein 36 (WDR36) on PDLSCs and found that WDR36 inhibited the migration, osteogenic differentiation, and senescence of PDLSCs. Thus, WDR36 may serve as a new candidate target for periodontal tissue regeneration.

INTRODUCTION

Periodontitis is an inflammatory disease caused by the host’s immune response and various interactions between pathogens, leading to the loss of connective tissue and bone[1,2]. The main pathological change of periodontitis involves the destruction of alveolar bone, which is the leading cause of tooth loss in adults[3]. Periodontitis also negatively impacts speech, nutrition, quality of life, and self-esteem[4-6]. Moreover, there is a risk of severe systemic inflammation[7,8]. Due to its high incidence, periodontitis has become a huge public health burden worldwide, and the treatment of periodontitis has become a significant health challenge. Thus, the ultimate treatment goal of periodontitis is to reconstruct the lost periodontal tissues with the same physiological structure and function[9].

Current therapies for periodontitis are not very effective for controlling inflammation or regulating immune function. It is especially difficult to achieve ideal regeneration of the lost periodontal tissue[10,11]. Tissue engineering/regenerative medicine is an established research field that combines biomaterials, cell therapy, biomedical engineering, and genetics[12]. Its purpose is to stimulate the regeneration of tissues and organs by implanting biomaterials in vivo or constructing substitutes in vitro[12]. Adult stem cells (SCs) have a high proliferation potential to differentiate into various cell types depending on the tissue of origin. Additionally, they are responsible for generating new tissues in response to injury and disease in vivo. Therefore, therapies based on adult SCs have received widespread attention in treating various degenerative diseases[13]. Notably, there are mesenchymal SCs (MSCs) in teeth and surrounding supporting tissues, which can be isolated and cultured in vitro and have the potential for self-renewal and multidirectional differentiation, as well as regeneration and repairing functions[10,11]. Moreover, MSCs can proliferate and differentiate to form new periodontal tissues[14-16]. Therefore, MSC transplantation technology is expected to be an ideal therapy for periodontal tissue regeneration.

Periodontal ligament SCs (PDLSCs) are among the few MSCs that continue to proliferate in adults. Additionally, their differentiation potential offers great promise for SC-based dental regeneration therapy[17,18]. PDLSCs are clonogenic and highly proliferative pluripotent cells that can regenerate cementum/periodontal ligament-like tissue by differentiating into osteoblast/cementoblast-like cells, adipocytes, chondrocytes, and fibroblasts. They also participate in the repair and steady-state turnover of periodontal tissue[19]. Previous studies have found that regenerated periodontal tissue can be obtained when autologous and allogeneic PDLSCs are transplanted into the periodontal defect area of pigs caused by surgery[16]. Therefore, PDLSCs are the leading candidate SCs among all MSCs, especially for treating periodontal tissue defects[20].

Research on traditional Chinese medicine has recently gained widespread attention, and this treatment method has unique advantages[21]. Icariin (ICA) is the main active flavonoid glycoside isolated from the dried stems and leaves of epimedium (chemical formula: C33H40O15); it has extensive pharmacological effects such as anti-oxidative and antitumor functions. ICA can dose dependently promote proliferation and differentiation, inhibit senescence, and reduce the apoptosis of PDLSCs[22-24]. ICA can also effectively promote bone reconstruction and prevent or delay the progression of bone metastasis-related diseases in rats by regulating the balance between adipogenesis and osteogenesis[23]. Our previous works showed that 0.1 μg/mL ICA could effectively promote proliferation and osteogenic differentiation and inhibit the bone resorption of PDLSCs[25,26]. To clarify the drug target of ICA, the whole-cell protein detection of PDLSCs under the action of 0.1 μg/mL ICA was performed. Western blotting verified the test results, and the target proteins and genes were subsequently screened. It was found that the expression of WD repeat-containing protein 36 (WDR36) was significantly increased.

Initially, WDR36 was discovered in 2005 as the candidate gene of primary open-angle glaucoma. The WDR36 gene is located in chromosome region 5q22.1, which spans a genomic region of approximately 34.7 kb and contains 23 exons that encode a 105 kDa protein (950 amino acids)[27]. WDR36 was found to be highly expressed in the human heart, placenta, liver, kidney, pancreas, and skeletal muscle[27]. By binding proteins, peptides, RNA, or DNA, the WDR domain participates in various cellular functions and is considered a possible drug target[28]. However, the role of WDR36 in MSCs and periodontal tissue regeneration remains unknown.

In this study, we used PDLSCs to clarify the function of WDR36 on the senescence, migration/chemotaxis, and osteogenic differentiation of PDLSCs and expound the potential role of WDR36 in periodontal tissue regeneration.

MATERIALS AND METHODS

Cell cultures

PDLSCs were purchased from Shanghai Optimal Ma Lai Biotechnology Co., Ltd. (Shanghai, China). The cells were cultured according to previously published protocols[29].

Cell sample preparation and proteomics analysis

ICA was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (No. 110737-200415; Beijing, China), with a purity of 98.5%. It is a yellow powder and was sealed and stored at 4 °C. Additionally, 1 mg ICA powder was dissolved in 2 mL phosphate-buffered saline (PBS) and prepared to a 0.5 g/L solution. Then the 0.5 g/L solution was diluted with Alpha Minimum Essential Medium [15% fetal bovine serum (FBS) + 2 mmol/L glutamine + 1% double antibody] to a mass concentration of 0.1 μg/mL ICA solution. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and cultured in ICA solution for 48 hours. Total protein extraction and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were performed according to previous protocols[30], followed by proteomics analysis according to previous protocols[31].

Plasmid construction and viral infection

The process of plasmid construction and viral infection was previously detailed in our study[29]. In short, plasmids were constructed using standard techniques, and all structures were verified through appropriate enzyme digestion and/or sequencing. Human full-length WDR36 cDNA containing the hemagglutinin (HA) tag was constructed using the whole gene synthesis method. Subsequently, the HA-WDR36 sequence was inserted into the pQCXIN recombinant vector through Agel and BamH1 restriction sites. Virus packaging was prepared according to the manufacturer’s protocol (Clontech Laboratories, Inc., Mountain View, CA, United States). Short hairpin RNA (shRNA)-WDR36 (sh-WDR36) and control shRNA lentivirus were purchased from Genepharma Company (Suzhou, China). The control shRNA sequence was 5’-TTCTCCGAACGTGTCACGT-3’, and the sh-WDR36 sequence was 5’-GCGATGGATTGCTCTATTAGG-3’.

Real-time reverse transcription polymerase chain reaction

RNA extraction, cDNA synthesis, and real-time reverse transcription polymerase chain reaction (RT-qPCR) procedures followed previous protocols[30]. RT-PCR was performed using the SYBR Green PCR kit (Qiagen, Hilden, Germany). All primer sequences are shown in Table 1. The 2^-ΔΔCt method was utilized to calculate the relative mRNA levels[32]. ΔCts were determined from the Ct normalized with GAPDH. Then we calculated the Pearson’s correlation coefficient for each gene using the normalized data to determine the consistency between microarray experiments and RT-qPCR (P < 0.05 and r > 0.9)[32].

Table 1 Real-time polymerase chain reaction primer sequence.

Gene	Primer sequences	Primer product size
GAPDH-forward	5’-CATGAGAAGTATGACAACAGCCT-3’	113 bp
GAPDH-reverse	5’-AGTCCTTCCACGATACCAAAGT-3’
WDR36-forward	5’-AGCCGTGGATGTTGTTGCTAT-3’	134 bp
WDR36-reverse	5’-GACCATCTGTGCGAAATGAAAT-3’
OSX-forward	5’-CTCCTGCGACTGCCCTAAT-3’	126 bp
OSX-reverse	5’-TGCGAAGCCTTGCCATAC-3’
RUNX2-forward	5’-CCAACCCACGAATGCACTATC-3’	78 bp
RUNX2-reverse	5’-CGGACATACCGAGGGACATG-3’
DLX3-forward	5’-CCAGACGGTGAACCCCTAC-3’	83 bp
DLX3-reverse	5’-CCGACTTGGGCGAGTAAGC-3’
OPN-forward	5’-ATGATGGCCGAGGTGATAGTGT-3’	76 bp
OPN-reverse	5’-TACTGGATGTCAGGTCTGCGA-3’
OCN-forward	5’-AAGAGACCCAGGCGCTACCT-3’	110 bp
OCN-reverse	5’-AACTCGTCACAGTCCGGATTG-3’
DMP1-forward	5’-GAAGAATGGAAGGGTCATTTGG-3’	134 bp
DMP1-reverse	5’-AAGCCACCAGCTAGCCTATAAATGT-3’

bp: Base pairs; DLX3: Distal-less homeobox 3; DMP1: Dentin matrix protein-1; OCN: Osteocalcin; OPN: Osteopontin; OSX: Osterix; RUNX2: Runt-related transcription factor 2.

Western blot analysis

Total protein extraction and SDS-PAGE were performed according to previous protocols[30]. The protein quantification used in the experiment was 20 μg. The primary antibodies used were anti-WDR36 (Cat No. 73548; Abcam, Cambridge, MA, United States) and mouse monoclonal anti-HA (Clone No. C29F4, Cat No. MMS-101P; Covance Laboratories, Inc., Princeton, NJ, United States). Membranes were probed with monoclonal antibodies against β-actin (Cat No. C1313; Applygen Technologies, Inc., Beijing, China) and GAPDH (Cat No. C1312-100/-250; Applygen), which served as loading controls.

Scratch-wound migration assays

PDLSCs were seeded into a 6-well plate (Corning Costar, Tewksbury, MA, United States) at 4-5 × 10⁵ cells/well and cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, United States) until the cells were 90% confluent. Then, a 1 mL pipette tip (Corning Costar) was used to scratch three straight wounds every 5 cm in the 6-well plate. After washing the cells with PBS (Gibco) to ensure no residual cell debris, the cells were cultured for 24-48 hours with DMEM. In sequence, three sites in each wound were selected to take pictures at 0 hour, 24 hours, and 48 hours. Image-Pro Plus 6.0 was applied to calculate the void area and height, and the relative width was obtained by the formula relative width = void area/height[33].

Transwell chemotaxis assays

To culture PDLSCs, 600 μL DMEM, supplemented with 15% FBS (Invitrogen, Carlsbad, CA, United States), was added to each well of the 24-well plate. Then the PDLSCs were seeded into the upper transwell chamber (8 μm pore size; Corning Costar) at 2-5 × 10⁴ cells/well and cultured for 24-48 hours under the conditions of 37 °C and 5% CO₂. Subsequently, the cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet staining solution. Finally, an inverted microscope and a camera system (Olympus, Tokyo, Japan) were used to observe the number of cells in nine randomly selected fields of each well.

Alkaline phosphatase activity assay and Alizarin red staining

PDLSCs were cultured in an osteogenic-inducing medium containing 100 μM/mL ascorbic acid (Cat No. abs580047; Absin, Shanghai, China), 2 mmol/L β-glycerophosphate (Cat No. 819-83-0; Kalamar, Shanghai, China), 1.8 mmol/L potassium dihydrogen phosphate [Cat No. GBW(E)130195; JissKang, Qingdao, China]_, and 10 nM dexamethasone (Cat No. A1217601; Gibco, Thermo Fisher Scientific, Shanghai, China). According to the manufacturer’s experimental procedures, alkaline phosphatase (ALP) activity was detected with an ALP Activity Kit (Sigma-Aldrich, St. Louis, MO, United States). After 14 days of osteogenic induction of PDLSCs, mineralization was detected by 70% ethanol fixation and 2% Alizarin red (Sigma-Aldrich) staining. Then the stained cells were destained with 10% cetylpyridinium chloride in 10 mmol/L sodium phosphate at room temperature for 30 minutes to determine the calcium content.

Preparation of ICA solution and senescence-associated β-galactosidase staining

The Senescence β-Galactosidase Staining Kit (Cell Senescence Testing Kit; GenMed Scientifics Inc., Shanghai, China) was used to detect the effects of ICA and WDR36 on the senescence of PDLSCs. The cells cultured in DMEM and cultured in ICA solution were washed three times with PBS and fixed in 4% paraformaldehyde at room temperature for 30 minutes. The cells were stained overnight with senescence-associated β-galactosidase (SA-β-gal) staining solution (GenMed Scientifics) at 37 °C and randomly selected from five different fields of view from each sample with an optical microscope. Following the selections, the percentage of blue cell was calculated from three independent experiments.

Statistical analyses

All statistical analyses were done using SPSS 16.0 statistical software (IBM SPSS Statistics, Armonk, NY, United States). The Student’s t-test or one-way analysis of variance was performed to evaluate the statistical significance, with P ≤ 0.05 considered statistically significant.

RESULTS

WDR36 expression increases with 0.1 μg/mL ICA

The results of cell proteomics analysis showed a significant increase in the expression of WDR36 (see the Supplementary material) after culturing PDLSCs with 0.1 μg/mL ICA for 48 hours. The results were further validated by Western blotting (Figure 1).

Figure 1 Expression of WD repeat-containing protein 36 in periodontal ligament stem cells with 0.1 μg/mL icariin. Western blot analysis showed that treatment with 0.1 μg/mL icariin (ICA) for 48 hours led to significantly increased expression of WD repeat-containing protein 36 (WDR36) in periodontal ligament stem cells (PDLSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control.

WDR36 overexpression inhibits the migration/chemotaxis of PDLSCs

The retroviral vector was used to construct the HA-WDR36 sequence and transfected into PDLSCs. After 7 days of screening with 600 μg/mL G418 antibiotic, the overexpression efficiency was verified by RT-qPCR and Western blotting (Figure 2). The 24-hour and 48-hour wound-healing results showed that the migration distance of the HA-WDR36 group was shorter than that of the control group. The difference was also statistically significant (P ≤ 0.01), indicating that WDR36 overexpression inhibited the migration function of the PDLSCs (Figure 3A and B). The transwell results also showed that the number of cells passing through the membrane in the vector group was significantly higher than that in the HA-WDR36 group (P ≤ 0.01), indicating that overexpression of WDR36 inhibited the chemotactic function of the PDLSCs (Figure 3C and D).

Figure 2 Validation of transfection efficiency of WD repeat-containing protein 36 overexpression. A: Reverse transcriptase-reverse polymerase chain reaction results showed the overexpression efficiency of hemagglutinin-WD repeat-containing protein 36 (HA-WDR36) in periodontal ligament stem cells (PDLSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control; B: Western blot analysis showed the overexpression efficiency of HA-WDR36 in PDLSCs. β-actin served as an internal control. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

Figure 3 Migration and chemotaxis of WD repeat-containing protein 36 overexpression. A and B: The results for 0 hour, 24 hours, and 48 hours showed that the cell migration distance of the control group was longer than that of the hemagglutinin-WD repeat-containing protein 36 (HA-WDR36) group; C and D: Transwell results showed that the number of cells passing through the membrane in the WDR36 overexpression group was less than that in the control group. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

WDR36 depletion promotes the migration/chemotaxis of PDLSCs

The sh-WDR36 lentivirus was used to knock down the expression of WDR36 in PDLSCs. After 3 days of screening with 1 μg/mL puromycin, the knockdown efficiency was verified by RT-qPCR and Western blotting (Figure 4). The 48-hour wound-healing results showed that the migration distance of the sh-WDR36 group was longer than that of the control group. The difference was also statistically significant (P ≤ 0.01), indicating that knocking down WDR36 promoted the migration function of the PDLSCs (Figure 5A and B). Transwell results showed that the number of cells passing through the membrane in the sh-WDR36 group was significantly higher than that in the control group (P ≤ 0.01), indicating that knocking down WDR36 promoted the chemotactic function of the PDLSCs (Figure 5C and D).

Figure 4 Validation of transfection efficiency of short hairpin RNA-WD repeat-containing protein 36. A: Reverse transcriptase-reverse polymerase chain reaction results showed the knockdown efficiency of short hairpin RNA-WD repeat-containing protein 36 (sh-WDR36) in periodontal ligament stem cells (PDLSCs). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control; B: Western blot results showed the knockdown efficiency of sh-WDR36 in PDLSCs. β-actin was used as an internal control. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05.

Figure 5 Migration and chemotaxis of short hairpin RNA-WD repeat-containing protein 36. A and B: The results for 0 hour and 24 hours showed that there were no significant differences between the short hairpin RNA-WD repeat-containing protein 36 (sh-WDR36) group and the control group. The 48 hours results showed that the cell migration distance of the control group was shorter than that of the sh-WDR36 group; C and D: Transwell results showed more cells passing through the membrane in the WDR36 depletion group than in the control group, and there was a statistically significant difference. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

WDR36 overexpression inhibits the osteogenic differentiation of PDLSCs

ALP activity was detected on the third and fifth days of osteogenic induction as an early indicator of osteogenic differentiation. The results showed that compared with the control group, the ALP activity of the experimental group was significantly decreased (P ≤ 0.05), demonstrating that the overexpression of WDR36 reduced the ALP activity of the PDLSCs. The results showed that WDR36 overexpression inhibited the early osteogenic differentiation of the PDLSCs (Figure 6A and B).

Figure 6 Osteogenic differentiation of WD repeat-containing protein 36 overexpression. A: Alkaline phosphatase (ALP) activity results on the third day; B: ALP activity results on the fifth day; C: Alizarin red staining; D: Calcium quantitative analysis; E: Expression of Runt-related transcription factor 2 (RUNX2) in the WD repeat-containing protein 36 (WDR36) overexpression group was significantly lower than that in the control group; F: Osterix (OSX) expression in the WDR36 overexpression group was significantly lower than that in the control group; G: Distal-less homeobox 3 (DLX3) in the WDR36 overexpression group was significantly lower than that in the control group; H: After 1 week and 2 weeks of osteogenic induction, osteopontin (OPN) expression in the WDR36 overexpression group was significantly lower than that in the control group; I: After 1 week of osteogenic induction, the expression of dentin matrix protein-1 (DMP1) in the WDR36 overexpression group was significantly lower than that in the control group. However, there were no statistically significant differences between the two groups at 0 week and 2 weeks; J: After 1 week of osteogenic induction, osteocalcin (OCN) expression in the WDR36 overexpression group was significantly lower than that in the control group. At 0 week and 2 weeks, no statistically significant differences between the two groups were observed. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05; ^bP ≤ 0.01.

On the 14^th day, the osteoblast-induced cells were stained with Alizarin red, and the calcium content was determined. We found that compared with the experimental group, the control group showed more obvious staining, and the calcium content of the control group was also higher than that of the experimental group. Notably, the results were statistically significant (P ≤ 0.05). The results showed that overexpression of WDR36 inhibited the mineralization of the PDLSCs in vitro (Figure 6C and D).

RT-qPCR was used to detect the expression of osteogenic factors Runt-related transcription factor 2 (RUNX2), osterix (OSX), and distal-less homeobox 3 (DLX3) in cells without osteogenic induction. The results showed that the expression of these osteogenic players in the WDR36-overexpressing group was significantly lower than that of the control group (P ≤ 0.05) (Figure 6E-G). After 1 week of osteogenic induction, the expression of osteopontin (OPN), dentin matrix protein-1 (DMP1), and osteocalcin (OCN) in the WDR36 overexpression group was significantly reduced compared with the control group (P ≤ 0.05) (Figure 6H-J). After 2 weeks of osteogenic induction, the expression of OPN in the experimental group was significantly lower than that in the control group (P ≤ 0.05) (Figure 6H).

WDR36 depletion promotes the osteogenic differentiation of the PDLSCs

The ALP activity results showed that compared with the control group, the ALP activity of the experimental group was significantly increased (P ≤ 0.05). This demonstrates that WDR36 depletion enhanced the ALP activity of the PDLSCs. That is, WDR36 depletion promoted the early osteogenic differentiation of the PDLSCs (Figure 7A and B).

Figure 7 Osteogenic differentiation of short hairpin RNA-WD repeat-containing protein 36. A: Alkaline phosphatase (ALP) activity results on the third day; B: ALP activity results on the fifth day; C: Alizarin red staining; D: Calcium quantitative analysis; E: Expression of Runt-related transcription factor 2 (RUNX2) in the WD repeat-containing protein 36 (WDR36) depletion group was significantly higher than that in the control group; F: Osterix (OSX) expression in the WDR36 depletion group was significantly higher than that in the control group; G: Distal-less homeobox 3 (DLX3) in the WDR36 depletion group was significantly higher than that in the control group; H: At 0 week and 1 week of osteogenic induction, the expression of dentin matrix protein-1 (DMP1) in the WDR36 depletion group was significantly higher than that in the control group. At 2 weeks, there were no statistically significant differences between the two groups; I: The difference in osteopontin (OPN) expression between the WDR36 depletion group and the control group was not statistically significant. However, after 1 week and 2 weeks of osteogenic induction, OPN expression in the depletion group was significantly higher than that in the control group; J: At 1 week of osteogenic induction, OCN expression in the WDR36 depletion group was significantly higher than that in the control group. However, there were no statistically significant differences between the two groups at 0 week and 2 weeks. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05; ^aP ≤ 0.01.

On the 14^th day, the osteoblast-induced cells were stained with Alizarin red, and the calcium content was detected. We found that compared with the experimental group, the control group showed less obvious staining, and the calcium content of the control group was also lower than that of the experimental group. Notably, these findings were statistically significant (P ≤ 0.05). The results show that knockdown of WDR36 promoted the mineralization of PDLSCs in vitro (Figure 7C and D).

The expression of osteogenic factors RUNX2, OSX, DLX3, and DMP1 in cells without osteogenic induction was detected by RT-qPCR. The results showed that expression in the sh-WDR36 group was significantly higher than that in the control group (P ≤ 0.05) (Figure 7E-H). After 1 week of osteogenic induction, the expression of DMP1, OPN, and OCN in the sh-WDR36 group was significantly increased compared with the control group (P ≤ 0.05) (Figure 7H-J). After 2 weeks of osteogenic induction, the expression of OPN in the experimental group was significantly higher than that in the control group (P ≤ 0.05) (Figure 7I).

ICA and WDR36 overexpression inhibits the senescence of PDLSCs

The results of SA-β-gal staining and quantitative calculation showed that the number of positive cells in the ICA group was significantly less than that in the group without ICA. In comparison, the number of positive cells in the HA-WDR36 group with or without ICA was significantly lower than that in the control group, indicating that 0.1 μg/mL ICA inhibited the senescence of PDLSCs. The same figure shows that overexpression of WDR36 also significantly inhibited the senescence of PDLSCs (P ≤ 0.01) (Figure 8).

Figure 8 Senescence of 0.1 μg/mL icariin and WD repeat-containing protein 36 overexpression. A: Senescence-associated β-galactosidase staining results; B: Senescence-associated β-galactosidase staining results showed that the number of positive cells in the icariin (ICA) group was significantly less than that in the group without ICA. By contrast, the number of positive cells in the hemagglutinin-WD repeat-containing protein 36 (HA-WDR36) group with or without ICA was significantly lower than that in the control group, which indicates that 0.1 μg/mL ICA inhibited the senescence of periodontal ligament stem cells (PDLSCs), and overexpression of WDR36 inhibited the senescence of PDLSCs. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^aP ≤ 0.05; ^bP ≤ 0.01.

WDR36 depletion promotes the senescence of PDLSCs

SA-β-gal staining results showed that the number of positive cells in the ICA group was significantly less than that in the group without ICA. In comparison, the number of positive cells in the sh-WDR36 group with or without ICA was significantly higher than that in the control group, indicating that 0.1 μg/mL ICA inhibited the senescence of PDLSCs. At the same time, WDR36 depletion promoted the senescence of PDLSCs (P ≤ 0.01) (Figure 9).

Figure 9 Senescence of 0.1 μg/mL icariin and short hairpin RNA- WD repeat-containing protein 36. A: Senescence-associated β-galactosidase staining results; B: Senescence-associated β-galactosidase staining results showed that the number of positive cells in the icariin (ICA) group was significantly less than that in the group without ICA. By contrast, the number of positive cells in the short hairpin RNA-WD repeat-containing protein 36 (sh-WDR36) group with or without ICA was significantly higher than that in the control group, which indicates that 0.1 μg/mL ICA inhibited the senescence of periodontal ligament stem cells (PDLSCs), and WDR36 depletion promoted the senescence of PDLSCs. The statistical significance was measured by the Student’s t-test. The error bars represent the standard deviation (n = 3). ^bP ≤ 0.01.

DISCUSSION

Our results indicate that WDR36 regulates the migration and chemotaxis, osteogenic differentiation, and senescence of PDLSCs. PDLSCs are the primary source of periodontal tissue regeneration, facilitating tissue repair by migrating to damaged sites. However, due to limited cell number and migration capacity, only a small number of PDLSCs can migrate to the target sites for periodontal tissue regeneration[33-35]. This could lead to less favorable conditions for subsequent periodontal regeneration. We investigated its effect on periodontal tissue regeneration by overexpression and knockdown of WDR36 in PDLSCs. The wound-healing and transwell results showed that overexpression of WDR36 inhibited the migration/chemotaxis of PDLSCs, whereas knocking down WDR36 promoted the migration/chemotaxis of PDLSCs. These findings indicate that WDR36 inhibits the migration/chemotaxis of PDLSCs, and WDR36 depletion is beneficial to the repair of damaged tissues.

We also studied the osteogenic differentiation function of PDLSCs by observing the osteogenic markers of cells at different times. Tissue non-specific ALP is generally considered a marker in the early process of osteogenesis/dentation and plays a role in bone matrix mineralization[36,37]. We detected the ALP activity and found that overexpression of WDR36 inhibited the ALP activity of PDLSCs while knockdown promoted its activity. The results also demonstrated that WDR36 inhibited the early osteogenesis of PDLSCs. Furthermore, by conducting Alizarin red staining and determining the calcium content, it was found that the overexpression of WDR36 inhibited the mineralization of PDLSCs in vitro. By contrast, WDR36 depletion enhanced the mineralization of PDLSCs in vitro.

We detected the mRNA expression of some osteogenic markers. The results showed that overexpression of WDR36 downregulated the expression of OCN, OPN, and DMP-1, suggesting that overexpression of WDR36 inhibited the osteogenic function of PDLSCs. OCN, OPN, and DMP-1 expression was upregulated in the WDR36 depletion group, indicating that knocking down WDR36 enhanced the osteogenic function of PDLSCs. RUNX2 is a transcription factor that regulates the transcription of many genes and early osteogenic differentiation. An increase in RUNX2 can promote osteoblast differentiation[38]. Moreover, OSX acts downstream of RUNX2, which maintains the balance of bone metabolism and is an important transcription factor for bone formation[39]. DLX3 plays a crucial role in embryogenesis and morphogenesis. Thus, the increased expression of DLX3 promotes the osteogenic function of MSCs[40]. Notably, our results showed that WDR36 overexpression decreased the expression of RUNX2, OSX, and DLX3 in PDLSCs. Meanwhile, WDR36 depletion increased their expression, suggesting that WDR36 inhibited the osteogenic differentiation of PDLSCs by regulating the transcription of RUNX2, OSX, and DLX3.

It has been shown that deleting the WDR36 gene in cultured human trabecular meshwork cells can eventually activate the p53 gene stress-response pathway, leading to cell apoptosis[41]. p53 can respond to various forms of cellular stress and is essential for cell senescence[42,43]. WDR36 is upregulated during human T-cell proliferation, participating in T-cell activation, and is considered a T-cell activation WD repeat protein[44]. However, research on WDR36 in osteogenic differentiation is still in its early stages, and the related mechanisms have not been investigated in the literature. Nonetheless, some works have found that the WD repeat protein, which activates gene transcription via chromatin remodeling, can accelerate osteoblast differentiation when overexpressed[45]. Furthermore, the gene WDR63, closely related to WDR36, is considered a key promoting factor in osteogenic differentiation[46]. The overexpression of WDR5 could enhance canonical Wnt signaling and osteoblast differentiation[45]. These investigations offer valuable insights and support for future works on the osteogenic differentiation mechanisms of WDR36.

ICA promotes the migration of bone marrow MSCs through hypoxia-inducible factor-1 alpha and further regulates the expression of chemokine receptor type 4[47]. ICA at a concentration of 0.01 mg/L effectively promotes the proliferation of PDLSCs and enhances the expression of osteoprotegerin (OPG) at both the mRNA and protein levels, thereby inhibiting alveolar bone resorption[25]. ICA can also promote the osteogenic differentiation of PDLSCs and stimulate the expression of osteogenic genes including ALP, OC, and type I collagen[25]. ICA promoted osteoblast proliferation by stimulating the expression of bone morphogenetic protein-2 (BMP-2) and OPG proteins and upregulating the mRNA expression of BMP-2, OPG, and ALP[23]. Some studies have found that ICA can promote the proliferation and differentiation of osteoblasts and induce the expression of RUNX2, OPG, and OSX through estrogen receptor-mediated pathways, BMP-2/Smad4 signaling pathways, and Wnt/β-catenin signaling pathways to increase the calcium content and promote bone formation[23,48,49]. Furthermore, ICA can downregulate the expression of RANKL through the nuclear factor kappa B and mitogen-activated protein kinases pathways to inhibit the proliferation, differentiation, and migration of osteoclasts, thus reducing bone resorption[50]. Our study revealed that ICA led to the increased expression of WDR36. However, it was found that the overexpression of WDR36 inhibited the migration and osteogenic differentiation of PDLSCs. This indicates that ICA and WDR36 exhibit antagonistic effects on osteogenic function. The contradictory results could be attributed to the negative feedback regulatory role of WDR36. The negative feedback loop is a crucial regulatory motif within cells that helps reduce the stochasticity of protein levels. Protein-mediated transcriptional regulation is a common form that has negative feedback mechanism, where the protein involved inhibits its own transcription[51]. Within the WD-repeat gene family, while both WDR63 and WDR5 exhibit promotion of osteogenic function[45,46], WDR7 and WDR8 both demonstrate inhibition of the osteogenic process[52,53]. The detected downregulation of WDR8 expression in the endochondral ossification process shows that the WDR8 might serve as an antagonist in endochondral ossification through a negative feedback regulatory mechanism[53].

Senescence is an inevitable physiological process of various organisms and the leading cause of many chronic diseases. The gradual weakening of the function of human organs characterizes it. With senescence, periodontal tissue’s anti-inflammation, anti-infection, and healing capacities gradually decrease. Additionally, the accumulated secretion of senescent cells may destroy tissue structure and function, affect SC function, cause environmental imbalance, and induce diseases[54]. MSCs also have anti-inflammatory, anti-fibrotic, and neuroprotective effects and can improve endothelial and mitochondrial functions, which makes MSCs attractive candidate cells for alleviating age-related diseases. Previous studies have also shown that ICA has antioxidant effects and can inhibit cell aging[13]. Treatment with 5 μM ICA during in vitro aging significantly reduced reactive oxygen species activity. Moreover, it increased the mRNA expression of glutathione and antioxidant genes. Additionally, 5 μM ICA prevented spindle defects and chromosome misalignment while increasing the mRNA expression of cytoplasmic maturation factor genes. It also inhibited apoptosis, increased the mRNA expression of anti-apoptotic genes, and decreased the mRNA expression of pro-apoptotic genes[22]. Therefore, we studied the effects of ICA and WDR36 on the senescence of PDLSCs. In 1995, Dimri et al[43] first proposed a marker enzyme to identify SA-β-gal, the product of the lysosomal β-gal gene beta-galactosidase (GLB1). In senescent cells, GLB1 significantly increases mRNA and protein levels, and the activity of β-gal is also correspondingly increased[30]. At a pH of 6.0, SA-β-gal can specifically recognize senescent cells in vitro and in vivo, and the positive staining rate increases with aging. Our results showed that the overexpression of WDR36 inhibited the senescence of PDLSCs, while the deletion of WDR36 promoted the senescence of PDLSCs. Simultaneously, we found that 0.1 μg/mL ICA inhibited the senescence of PDLSCs. Based on these findings, it was found that while WDR36 and ICA exhibit opposing functions in osteogenic process, they demonstrated a synergistic effect in terms of senescence-related functions. Although the negative feedback of WDR36 counteracted some anti-senescence effects, overexpression of WDR36 with ICA still exhibited some anti-senescence functions in PDLSCs. Previous investigations have also confirmed that the upregulation of WDR7 in rat PDLSCs could exert protective effects against hydrogen peroxide-induced oxidative stress, which is a significant factor contributing to cellular aging[55,56]. However, more works on the feedback mechanism of WDR36 are needed, such as the feedback regulatory pathways of WDR36 and the identification of key regulators within these pathways. These could reduce the negative impact of the feedback regulation on osteogenesis and anti-aging functions, and provide the possibility for our research to be applied clinically in the treatment of periodontal diseases in the future.

Although some studies have demonstrated the regulatory role of WD-repeat protein in osteogenic differentiation[45], research on WDR36 in osteogenesis is still lacking. As such, our study has addressed this research gap. Presently, the treatment of periodontitis primarily involves medication, basic therapy, surgical interventions, and supportive care. However, the effectiveness of these treatment modalities is not sufficiently ideal, especially in achieving optimal regeneration of lost periodontal tissues. PDLSCs can potentially promote the regeneration of periodontal tissues, while ICA can dose dependently stimulate the proliferation, differentiation, and deceleration of senescence of PDLSCs. Through our work, we demonstrated that targeting WDR36 effectively regulates the osteogenic differentiation of PDLSCs. This finding could introduce a novel approach to the regenerative treatment of periodontitis.

CONCLUSION

In conclusion, WDR36 has regulatory effects on PDLSCs. As such, it inhibits the migration and chemotaxis, osteogenic differentiation, and senescence of PDLSCs. 0.1 μg/mL ICA inhibits the senescence of PDLSCs. Thus, our results provide new ideas and candidate targets for periodontal tissue regeneration and the treatment of periodontitis.

ACKNOWLEDGEMENTS

The authors gratefully thank the reviewers for their improvements to the manuscript.